RU2534822C2 - Method of estimating pharmacological and toxicological properties of substances - radio-, toxicoprotectors and radio-, toxicosensibilisers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of radiobiology and experimental medicine. A method of estimating pharmacological and toxicological properties of substances consist in the following: a substance to be analysed is introduced into a nutritional medium of larvae and flies of Drosophila melanogaster, combining in their genome hypomorphic mutations of ss- and CG5017-genes. The larvae and flies are irradiated with ionising rays with a dose of 1-10 roentgen. Viability, structures of extremities and a level of transcription of CG 1681, CYP6G1 and ss-genes are estimated. The obtained characteristics of the flies, grown on a medium, containing the analysed substance, and the flies, grown on a medium, which does not contain the analysed substance, irradiated and non-irradiated are compared, and pharmacological properties of the substance are determined by the results of comparison of viability, quantity of tarsal segments of extremity and the level of transcription of CG 1681, CYP6G1 and ss-genes in the flies of all formed groups.

EFFECT: method makes it possible to realise effective fast targeted selection and determine the properties of substances with toxicoperotective, radioprotective, toxicosensibilising and radiosensibilising properties.

7 dwg, 2 tbl, 2 ex

Description

The impact of various types of ionizing radiation on a person in everyday life is quite large. Such risks arise in the daily lives of many people. In particular, for carriers of hypomorphic mutations of homologous genes in humans, visits to the X-ray room, long-term flights on airplanes, work on nuclear reactors, prolonged exposure to high concentrations of radon (in basements), etc. can be dangerous. In addition, with the increase in human age due to the influence of somatic mutagenesis, the number of people for whom the level of danger of ionizing radiation is increased.

The present invention relates to research in the field of biology and experimental medicine and can be used in biomedical research, in particular toxicological, radiological, pharmacological, biophysical studies and for laboratory and experimental assessment and targeted search of substances - radio and toxic protectors with the properties of radio , toxicoprotection and radio-, toxicosensitization and contributing to the adaptive response of the body to the toxic effects of various types of radiation sludge exogenous toxins. Traditionally, for the identification, evaluation and directed search for the pharmacological, toxicological, pharmaceutical properties of substances - radio and toxic protectors by their ability to induce a metabolic response to the toxic effects of various species, including the effects of ionizing radiation, various model objects of laboratory animals - mice, rats - are used , rabbits, dogs and their cells bred in culture. These objects are evolutionarily close to humans, but the wide range of available genetic methods and the high cost of research limits the possibility of their use.

Closest to the claimed method is a technical solution according to the patent KR 20120020820 (A) - SCREENING METHOD OF A TOXIC MATERIAL POLLUTING INTERIOR ATMOSPHERE USING DROSOPHILA MELANOGASTER [1], which proposes a research method using mutant Drosophila lines for determination of harmful substances in indoor air buildings. The basis of the proposed patent is the use of microchips to identify genes activated by exposure to gaseous toxins inhaled by flies. To do this, it is proposed to use Drosophila lines mutant in genes whose protein products are involved in the direct process of metabolic response to the action of various kinds of toxins. The list of mutations proposed for use includes genes encoding: heat shock proteins, glutathione-S-transferase, cytochrome P450, superoxide dismutase, catalase and a number of other proteins that work at different phases of neutralizing the effects of various toxins. The method with high sensitivity proposed in [1] makes it possible to detect the presence of harmful substances in the air of the interior of buildings. However, this method does not involve the search for substances capable of modifying the expression of genes that regulate the metabolic response to the action of harmful substances during in vivo development and which can reduce or increase the risk of developmental disorders.

The present invention is based on the technical task of creating an effective, directed, reliable and highly sensitive method for studying and evaluating the pharmacological and toxicological properties of substances with putative toxicoprotective, radioprotective, toxicosensitizing and radiosensitizing properties that can reduce or increase the toxicity of ionizing radiation, for the selection of such substances and further use in creating pharmaceuticals that can influence on the toxic effects of radiation and other exogenous and endogenous factors.

The generally accepted requirements for evaluating the effects of radiation on organisms involve the use of isogenic lines in laboratory animals that do not have mutations in their genome for genes involved in the metabolic regulation of the response to radiation. These primarily include genes whose expression is aimed at normalizing the level of superoxide compounds, an increase in which is one of the consequences of ionizing radiation. An important element of the response of mammalian cells to an increase in the level of superoxide compounds is the activation of AHR gene expression. Its protein product is the Aryl Hydrocarbon Receptor (AHR), a receptor for many toxins of both exogenous and endogenous origin. One of the endogenous AHR ligands is a toxic derivative of tryptophan - formyl-indole-carbazole (FICZ). The level of FICZ concentration in cells increases significantly in response to ionizing radiation. This stimulates an increase in AHR activity. Reports of the participation of AHR in the reaction to the effects of ionizing radiation and the impaired processes of neurogenesis and memory caused by it give rise to fears of a stronger effect of ionizing radiation and other toxic factors on people who have hypomorphic mutations of the AHR gene in their genome [2], [3] . With increasing age, somatic mutagenesis expands the number of people for whom the level of danger of ionizing radiation is increased. Such risks arise in the daily lives of many people. Despite the fact that the risks are primarily indicated for humans, the high conservatism of the structure and functions of the AHR gene and its targets allows you to take the first steps in evaluating substances that can enhance or attenuate the toxic effects of radiation using Drosophila as a test system.

In Drosophila, the manifestation of the ss ^a40a hypomorphic mutation in the spineless gene, which is a homologue of the AHR gene, is sharply enhanced if it is combined in one genome with hypomorphic mutations of other genes involved in the formation of memory, reaction to heat shock, and cell polarization. Using the example of a double mutant carrying the hypomorphic ss ^a40a mutation and the CG5017 gene mutation, we can demonstrate the effects on morphogenesis and on the adaptive response caused by ionizing radiation of individuals combining two hypomorphic mutations in one genome.

Mutations of the spineless gene to varying degrees are capable of disrupting: bristle morphogenesis; distal structures and antennas and limbs; dendritic structures of neurons and photoreceptors.

The CG5017 gene encodes a nucleotropic chaperone belonging to the NAP 1 protein family. Mutation of the CG5017 gene does not violate morphogenesis, but causes a violation of the formation of long-term memory [4]. Taking into account the molecular functional features of ss and CG5017 gene products and the high degree of conservatism of their expression products, one can quite reasonably rely on the results of a study of their interaction to assess the risks of combining mutations in the homologs of these genes in humans and the possibilities of their pharmacological correction. At the same time, it can be assumed that even weak mutations of such genes can alter the body's adaptive response to the action of ionizing radiation or other toxic agents. Since human populations as a result of the created social conditions of modern society are burdened with many hypomorphic mutations, the probability of meeting one or more hypomorphic mutations in the same genome is quite high. Their carriers can safely exist, but for them, the effects of radiation and other toxic agents pose an increased risk even in cases of small doses.

To enhance the demonstration of the effect of the interaction of hypomorphic mutations, a specially selected hypomorphic subline of ss a40ahm flies was ^used , which differs from the ss ^a40a original line ^{flies by a} less pronounced transformation of the distal parts of the antenna into tarsus and the CG5017 mutant locus, milk a h flies, which have no morphological abnormalities, but differ long-term memory [4].

Hybrid flies ss ^a40ahm CG5017 (milk a h) differ from ss ^{a40ahm in a} sharply enhanced manifestation of a mutant ss phenotype (Fig. 1). Figure 1 shows the amplification of the mutant phenotype in hybrid flies ss ^a40ahm milk a h, which manifests itself in an increase in the homeosis transformation of the distal antenna structures into tarsus and in violation of limb segmentation.

In hybrid flies, an increase in the manifestation of the ss phenotype is observed not only in antenna structures, but also in the tarsal segments of the limbs (Table 1-2, figure 2). Their phenotype was consistent with the phenotype of ss ^aSc flies obtained in independent experiments as a result of crossing flies from the super mutagenic, dysgenic line w oc / FM4 with flies from the ss ^a40ahm line. In addition to the mutation at the ss locus, ss ^aSc flies have an insertion of the P element in the region of the CG5017 gene, which significantly suppresses its expression level [5]. However, the manifestation of the mutant ss phenotype in flies combining mutations at the loci of ss and CG5017 genes is even more intensified after irradiation of larvae of the third developmental age (Tables 1–2, Fig. 2).

In figure 2 and from table 1-2 it is seen that the combination in the genome of two mutations at once in the spineless and CG5017 loci makes the body sensitive even to low radiation doses of 1-10 R, which is manifested in the amplification of the mutant phenotype. It is important to note that the degree of mutant manifestation of the ss phenotype also depends on the saturation of the nutrient medium with toxic waste products of Drosophila larvae. This means that, by introducing certain substances into the nutrient medium, one can study their influence on the development both in the direction of the protection of ionizing radiation and in the direction of increasing the risk of its exposure. This effect can be estimated by the qualitative and quantitative indicators of the homeosis transformation of antenna structures and the violation of tarsal segmentation. The presented results and illustrations illustrating them, together with a discussion, are given to facilitate the use of this technical solution.

Another important indicator of the effect of pharmacological agents on AHR can be an assessment of their effect on the level of expression of CYP genes (encode proteins of the P450 cytochrome family) and GST (encode proteins of the glutathione S transferase family). These genes are involved in the metabolic response of cells to the toxic effects of superoxide compounds, a sharp increase in the level of which is the basis of the toxic effects of ionizing radiation on cells. The level of their expression changes at different phases of the metabolic response of cells to the toxic effect of superoxide compounds. Below is shown the change in the expression level of CG1681 - (belongs to the family of GST genes) and CYP6G1 genes in response to x-ray irradiation in flies of normal genotype and in flies combining mutations in ss and CG5017 loci in their genome. The structure of both genes contains target 8 nucleotide CRE recognition motifs for AHR. The results of measuring the level of transcription of CYP6G1 CG1681 and ss in irradiated flies, obtained using the polymerase chain reaction method, in real time are presented in figure 3 in the form of graphs. Depicted in Figure 3 graphs show an increase transcription levels CG1681- CYP6G1-genes and the wild type C a nton S flies in response to x-irradiation, even in small doses in the range 1-10R. Irradiation of ss ^aSc flies, combining mutations in the ss and CG5017 loci in their genome, leads to a decrease in the level of transcripts of CYP6G1 and CG1681 genes. AHR deficiency in these flies does not provide compensatory regulation of transcription of CYP6G1 and CG1681 genes, the products of which are in demand during biodegradation of radiotoxins. Considering the fact that morphological abnormalities after irradiation occur only in ss ^aSc flies (Tables 1–2, Fig. 2), it can be concluded that their lack of compensatory regulation of transcription of CG1681-, CYP6G1-, and ss-genes causes disturbances morphogenesis of limbs. Differences in the consequences of x-ray irradiation for line flies and ss ^aSc indicate an increased risk of implementing pathology development scenarios in humans, combining various hypomorphic gene mutations in their genome. The used lines of flies contain mutations of highly conserved genes, leading to developmental and metabolic disorders according to scenarios similar to both a fly and a human. Therefore, it is proposed to use the Drosophila fly line, combining hypomorphic mutations of ss and CG5017 genes in its genome, as a sensitive test system for the targeted search for pharmaceuticals with toxic protective, radioprotective, toxic sensitizing and radiosensitizing properties that can affect the toxic effects of radiation and and endogenous factors. Pharmaceutical agents with toxicoprotective, radioprotective, toxicosensitizing and radiosensitizing properties found with its help may also be in demand in gerontology, since with age, a person increases the risk of mosaic occurrence of somatic mutations.

The following examples describe the procedures necessary to assess the ability of substances to enhance or weaken the effect of a toxic agent by their ability to correct the development of distal limb structures and the level of transcription of CG1681 and CYP6G1 genes in Drosophila flies combining hypomorphic mutations of ss and CG5017 genes in their genome .

Tables 1 and 2 show the incidence of extremities with normal (five) and abnormal (less than five) the number of tarsal segments and the survival (death) of wild-type flies and double ss ^a40a CG5017 mutants under normal conditions and on medium with furazidine (Table. 1), serotonin (Table 2) and upon irradiation (1-10 R).

Figure 1 shows an example of the formation of distal structures of the antenna and extremities in wild-type flies - C a nton S and mutant lines: ss ^a40ahm , milk a h, ss ^a40ahm milk a h;

Figure 2 presents an example of enhancing radiation sensitivity in carriers of a hypomorphic mutation in the ss gene when combining it with a mutation in the CG5017 gene;

Figure 3 presents the results of measuring the level of transcription of the CYP6G1, CG1681 and ss genes in flies before and after irradiation.

Figure 4 (see examples) presents the frequency of occurrence of limbs with a different number of tarsal segments in double ss ^a40a CG5017 mutants when growing larvae on medium with furazidine. The green bar (5 segments) characterizes the normal morphology of the limb.

Figure 5 (see examples) presents the results of measuring the level of transcription of the CYP6G1, CG1681 and ss genes in irradiated flies under the conditions of their larvae on standard feed medium and a medium containing furazidine.

Figure 6 (see examples) shows the frequency of occurrence of limbs with a different number of tarsal segments in double ss ^a40a CG5017 mutants when growing larvae on a medium with serotonin. The green bar (5 segments) characterizes the normal morphology of the limb.

Figure 7 (see examples) presents the results of measurements of the level of transcription of the CYP6G1, CG1681 and ss genes in irradiated flies under conditions of their larvae on standard feed medium and a medium containing serotonin.

Example 1. Description of procedures aimed at assessing the properties of furazidine (Furazidin - a derivative of nitrofuran compounds) by the ability to correct disturbances in the development of distal structures of adult limbs and compensatory regulation of the expression level of CG1681- (belongs to the family of GST genes) - and CYP6G1 genes in response to X-ray irradiation of Drosophil a mel a nog a ster.

To assess the ability of furazidine to correct the effects of radiation on the development of limb structures and the viability of developing flies, 600-800 pairs of males and females of the CS line (wild line) and ss ^aSc (combining hypomorphic mutations in the ss and CG5017 gene loci in their genome) were selected Drosophil a mel a nog a ster. The selected flies of each line were placed in a separate 0.5 liter wide-necked bottle with a diameter of 30-40 mm (150-200 pairs of flies in one bottle). In addition to the throat hole, the bottle had a ventilation hole covered with cotton wool. A Petri dish filled with standard feed medium of the following composition was inserted into the neck of the bottle: 1% agar, 10% sucrose, 3% dry yeast, 8% cornmeal, 28% grape wine or juice, 0.002% mold inhibitor, 50% water. A day later, a Petri dish with eggs laid in each bottle was changed to another with fresh feed medium. The surface of the medium with laid eggs was freed from hatching larvae. After a time period of 1-2 hours, newly hatched larvae were selected (at least 200 individuals) and transferred 25-30 larvae into test tubes with 10 milliliters of fresh standard nutrient medium. The larvae transferred to test tubes developed at a temperature of 25 ° C for two days. After two days of the second molt of the third age, the larvae of the third age were transferred to freshly prepared standard nutrient medium (Carolina Biological Sapply - Formula 4-24) in two tubes (25-30 larvae each) and with additional 80 mg / l furazidine added in two test tubes (25-30 larvae each). After another day, half of the larvae in each group were irradiated with X-rays at a dose of 1-10 R. An RUM-17 X-ray machine was used as the radiation source. Thus, eight groups of larvae were formed during the study:

1 - a group of larvae of normal genotype (CS), developed on a standard medium, not exposed to x-ray irradiation;

2 - a group of ss ^aSc larvae that developed on a standard medium and were not exposed to x-ray irradiation.

3 - a group of larvae of normal genotype (CS), developed on a standard medium, subjected to x-ray irradiation;

4 - a group of ss ^aSc larvae that developed on a standard medium and were exposed to x-ray irradiation;

5 - a group of larvae of normal genotype (CS), developed on a standard medium containing furazidine at a concentration of 80 mg / l, not exposed to x-ray irradiation.

6 - a group of ss ^aSc larvae that developed on a standard medium containing furazidine at a concentration of 80 mg / L, not exposed to x-ray irradiation;

7 - a group of larvae of normal genotype (CS), developed on a standard medium containing furazidine at a concentration of 80 mg / l, subjected to x-ray irradiation;

8 - a group of ss ^aSc larvae that developed on a standard medium containing 80 mg / l furazidine, subjected to x-ray irradiation.

After completion of the larval development process and metamorphoses of all adults, they were collected, fixed in ethyl alcohol and the structures of the extremities were analyzed, assessing the degree of transformation of the arista into tarsus and the number of tarsal limb segments. The properties of furazidin were evaluated by comparing the structures of 100 extremities of flies of each group, wild and mutant, grown on a medium containing furazidine and grown on standard nutrient medium without the addition of furazidine, irradiated and non-irradiated by the number of tarsal limb segments in flies of each group. The results of evaluation of properties furazidina abilities to modify the radiation effect on the development of limbs flies line ^{ss aSc Drosophil a mel a nog a} ster, combining mutations in its genome loci CG5017-ss- and genes shown in Table 1 and Figure 4. The presented results indicate the radiosensitizing properties of furazidine. This is indicated by the 70% death of ss ^aSc larvae subjected to the combined action of radiation and furazidine. At the same time, the tarsal segmentation of ss ^aSc flies surviving after such exposure is significantly normalized. In 80% of the surviving flies, the tarsus had 4 segments. This can be explained by incomplete synchronization of the development of the larvae involved in the experiment. For some of them, irradiation occurred in the least radiosensitive period of development.

To assess the ability to adjust furazidina effects of radiation on the ability to influence the level of transcription CG1681-, CYP6G1- and ss- genes collected on 240 male adults of lines and CS ^{ss aSc Drosophil a mel a nog a} ster one-day age.

From the selected males of each line, 4 groups of 60 flies were formed in each group.

The first group, the CS line, was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24). After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The second group, the ss ^aSc line, was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24). After 30 hours, the flies were frozen and crushed in liquid nitrogen.

Group 3 — CS — was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply — Formula 4-24) and exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The fourth group, ss ^aSc , was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The 5th group - CS - was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing furazidine at a concentration of 80 mg / L. After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The 6th group - ss ^aSc - was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing furazidine at a concentration of 80 mg / L. After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The 7th group, CS, was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing furazidine at a concentration of 80 mg / l, and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The 8th group - ss ^aSc - was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing furazidine at a concentration of 80 mg / L, and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

Total RNA was isolated from frozen and ground in liquid nitrogen flies using Tri Reagent (Sigma, USA) in accordance with the manufacturer's recommendations. Tri Reagent is added to the samples ground in liquid nitrogen at the rate of 1 ml per 100 mg of sample. Then centrifuged for 10 min at 10,000 g. Then, chloroform was added to the supernatant at the rate of 0.2 ml of chloroform per 1 ml of TriReagent. Shaken at high speed for 15 seconds. Next, the resulting mixture was incubated for 10 min at room temperature. Then, the resulting mixture was centrifuged at 12000 g for 15 minutes. The supernatant was taken and phenol-chloroform extraction was performed. Next, RNA was precipitated in the presence of isopropanol at the rate of 0.5 ml of isopropanol per 1 ml of TriReagent used. The resulting RNA pellet was washed with 75% ethanol 3 times. The precipitate was dried and dissolved in an appropriate volume of sterile nuclease-free water. All centrifugations were carried out at 4 ° C. The quality of the obtained RNA was checked in 1% agarose in the presence of ethidium bromide. For electrophoresis, RNA was mixed with gel buffer (Formaldehyde loading dye, Ambion, USA) at the rate of 3 buffer volumes per volume of RNA solution. Then, the resulting mixture was incubated for 3 min at 70 ° C, after which the resulting mixture was incubated in ice for 5 min. Then it was applied to the gel.

In order to assess the level of transcription of the ss, CG1681 and CYP6G1 genes, the real-time NDP (PCR-RV) method was used. When working on the invention, an ABI PRISM® 7500 Sequence Detection System, Applied Biosystems, USA was used. Each reaction was repeated 3 times in a volume of 25 μl. In the work, a comparative, or ddCt, method is used, which allows one to evaluate changes in the mRNA level with a double comparison: of the studied gene with a control gene between different samples. Reactions were carried out in relative measurement mode (RQ-relative quantification software, Applied Biosystems, USA). For the reactions, the prepared reaction mixtures Synthol (Russia) were used. Reaction conditions: 10 min at 95 ° C (“hot start” to activate DNA polymerase) and then 40 cycles: 15 sec at 95 ° C (denaturation) and 1 min at 60 ° C (annealing with primers and elongation) . Standard curves i.e. curves of CT versus decimal logarithm of the amount of added cDNA (log C0) for the studied cDNA sequences and calculation of amplification efficiencies for each sequence were calculated using ABI Prism 7500 SDS Software Version 2.0.3 (Applied Biosystems, United States). Real-time PCR results were analyzed using ABI Prism 7500 SDS Software Version 2.0.3 (Applied Biosystems, USA) in accordance with the recommendations of Applied Biosystems. For the reactions used 10 pcM and 10 pcM primers and linear destructible probe (TaqMan®), respectively. The gene of ribosomal protein Rpl32 was used as a control gene: The following pairs of primers and probes were used for amplification:

for the Rpl32 gene: primers Rpl32dir 5′-CCAGCATACAGGCCCAAGATC-3 ′, Rpl32rev 5′-ACGCACTCTGTTGTCGATACC-3 ′, probe - FAM-CGCACCAAGCACTTCATCCGCCAC-BHQ1;

for the Cyp6g1 gene: primers Cyp6g1f 5′-GCGATCCATTGGGCTATAAT-3 ′, Cyp6g1r 5′-CCAATCTCCTGCATAAGGGT-3 ′, probe - FAM-TCGCACCAAGCTGACTCCCG-BHQ1;

for gene CG1681 (GST-thet a ): primers CG1681f 5′-TTCGCACCCACTCTCTAGTCAC-3 ′, CG1681r 5′-GCTCGATTGGTTCAGGAAAT-3 ′, probe - FAM-TCAACGAGATGTCGCAGCCACTC-BHQ1;

for the ss gene: primers SSdir 5′-CGAAGGCGACGCAACGG-3 ′, SSrev 5′-GATGCCGCTTTGATGGATTGC-3 ′, probe — FAM-AGCCTGAAGCCGCCGCCCAAG-BHQ1.

In our experiments, probes were synthesized in Syntol (Russia), oligonucleotide primers in Litech (Russia).

The analysis of PCR-PB products was carried out on a 2% agarose TAE gel. All amplification products had the expected size.

The results of the analysis of the level of transcription of genes CG1681, CYP6G1 and ss in the analyzed flies are presented in figure 5.

A comprehensive assessment of the properties of furazidine allows you to attribute it to toxic substances with toxicosensitizing and radiosensitizing effects. The reason for this is the death of the overwhelming majority of ss ^aSc larvae recorded by us when the action of furazidine and X-ray irradiation on them was combined (Table 1). The action of furazidine causes a slight increase in the level of transcription of CG1681-, CYP6G1- and ss-genes. This is enough to normalize the tarsal segmentation in surviving flies, but not enough to neutralize the toxic effect.

Example 2. Description of procedures aimed at assessing the properties of 5-hydroxytryptamine (serotonin) at a concentration of 80 mg / l by the ability to correct impaired development of distal structures of the adult limbs and compensatory regulation of the expression level of CG1681- (belongs to the family of CST genes) - and CYP6G1- genes in response to X-ray irradiation of Drosophil a mel a nog a ster.

To assess the ability of the properties of 5-hydroxytryptamine to correct the effects of radiation on the development of limb structures and the viability of developing flies, 600-800 pairs of males and females of the CS line (wild line) and ss ^aSc (combining hypomorphic mutations at the ss- and CG5017 loci in their genome) were selected genes) Drosophil a mel a nog a ster. The selected flies of each line were placed in a separate 0.5 liter wide-necked bottle with a diameter of 30-40 mm (150-200 pairs of flies in one bottle). In addition to the throat hole, the bottle had a ventilation hole covered with cotton wool. A Petri dish filled with standard feed medium of the following composition was inserted into the neck of the bottle: 1% agar, 10% sucrose, 3% dry yeast, 8% cornmeal, 28% grape wine or juice, 0.002% mold inhibitor, 50% water. A day later, a Petri dish with eggs laid in each bottle was changed to another with fresh feed medium. The surface of the medium with laid eggs was freed from hatching larvae. After a time period of 1-2 hours, newly hatched larvae were selected (at least 200) and transferred in an amount of 25-30 larvae into test tubes with 10 mg of fresh standard nutrient medium. The larvae transferred to test tubes developed at a temperature of 25 ° C for two days. After two days of the second molting of third age larvae, they were transferred to freshly prepared standard nutrient medium (Carolina Biological Sapply - Formula 4-24) in two tubes (25-30 larvae each) and with additional 5-hydroxytryptamine at a concentration of 80 mg / l in two test tubes (25-30 larvae each). After another day, half of the larvae in each group were irradiated with X-rays at a dose of 1-10 R. An RUM-17 X-ray machine was used as the radiation source. Thus, eight groups of larvae were formed during the study:

1 - a group of larvae of normal genotype (CS), developed on a standard medium, not exposed to x-ray irradiation;

2 - a group of ss ^aSc larvae that developed on a standard medium and were not exposed to x-ray irradiation.

3 - a group of larvae of normal genotype (CS), developed on a standard medium, subjected to x-ray irradiation;

4 - a group of ss ^aSc larvae that developed on a standard medium and were exposed to x-ray irradiation;

5 - a group of larvae of normal genotype (CS), developed on a standard medium containing serotonin at a concentration of 80 mg / l, not exposed to x-ray irradiation;

6 - a group of ss ^aSc larvae that developed on a standard medium containing serotonin at a concentration of 80 mg / l, not exposed to x-ray irradiation;

7 - a group of larvae of normal genotype (CS), developed on a standard medium containing serotonin at a concentration of 80 mg / l, subjected to x-ray irradiation;

8 - a group of ss ^aSc larvae that developed on a standard medium containing serotonin at a concentration of 80 mg / L, subjected to x-ray irradiation;

After completion of the larval development process and metamorphoses of all adults, they were collected, fixed in ethyl alcohol and the structures of the extremities were analyzed, assessing the degree of transformation of the arista into tarsus and the number of tarsal limb segments. The properties of serotonin were evaluated by comparing the structures of 100 extremities of flies of each group, wild and mutant, grown on a medium containing serotonin, and grown on standard nutrient medium without the addition of 5-hydroxytryptamine, irradiated and non-irradiated by the number of tarsal limb segments in flies of each group. The results of evaluation of properties of serotonin by the ability to modify the radiation effect on the development of limbs flies line ^{ss aSc Drosophil a mel a nog a} ster, combining mutations in its genome loci ss- and CG5017-genes are presented in Table 2 and Figure 6.

To assess the ability to adjust the properties of serotonin effects of radiation on the ability to influence the level of transcription CG1681-, CYP6G1- and ss-genes in the fly line ^{ss aSc, Drosophil a mel a nog} a ster, combining its genome hypomorphic mutations at loci and ss- CG5017 genes, 240 male adults of the CS and ss ^aSc Drosophil a mel a nog a ster lineage of one day old were collected.

Selected males Drosophil a mel a nog a ster divided into groups of 60 flies.

The first group, CS, was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24). After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The second group - ss ^aSc - was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24). After 30 hours, the flies were frozen and crushed in liquid nitrogen.

Group 3 — CS — was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply — Formula 4-24) and exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The fourth group, ss ^aSc , was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The 5th group - CS - was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing serotonin at a concentration of 80 mg / L. After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The 6th group, ss ^aSc , was transferred into a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing serotonin at a concentration of 80 mg / L. After 30 hours, the flies were frozen and crushed in liquid nitrogen.

The 7th group, CS, was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply - Formula 4-24) containing serotonin at a concentration of 80 mg / L, and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

The 8th group, ss ^aSc , was transferred to a plastic tube with 10 mg of fresh nutrient medium (Carolina Biological Sapply — Formula 4-24) containing serotonin at a concentration of 80 mg /, and was exposed to x-rays a day later. 6-7 hours after exposure to radiation, the flies were frozen and crushed in liquid nitrogen.

Total RNA was isolated from frozen and ground in liquid nitrogen flies using Tri Reagent (Sigma, USA) in accordance with the manufacturer's recommendations. Tri Reagent is added to the samples ground in liquid nitrogen at the rate of 1 ml per 100 mg of sample. Then centrifuged for 10 min at 10,000 g. Then, chloroform was added to the supernatant at the rate of 0.2 ml of chloroform per 1 ml of TriReagent. Shaken at high speed for 15 seconds. Next, the resulting mixture was incubated for 10 min at room temperature. Then, the resulting mixture was centrifuged at 12000 g for 15 minutes. The supernatant was taken and phenol-chloroform extraction was performed. Next, RNA was precipitated in the presence of isopropanol at the rate of 0.5 ml of isopropanol per 1 ml of TriReagent used. The resulting RNA pellet was washed with 75% ethanol 3 times. The precipitate was dried and dissolved in an appropriate volume of sterile nuclease-free water. All centrifugations were carried out at 4 ° C. The quality of the obtained RNA was checked in 1% agarose in the presence of ethidium bromide. For electrophoresis, RNA was mixed with gel buffer (Formaldehyde loading dye, Ambion, USA) at the rate of 3 buffer volumes per volume of RNA solution. Then, the resulting mixture was incubated for 3 min at 70 ° C, after which the resulting mixture was incubated in ice for 5 min. Then it was applied to the gel.

To assess the level of transcription of the CG1681 and CYP6G1 genes, the real-time PNR method (PCR-RV) was used. When working on the invention, an ABI PRISM® 7500 Sequence Detection System, Applied Biosystems, USA was used. Each reaction was repeated 3 times in a volume of 25 μl. In the work, a comparative, or ddCt, method is used, which allows one to evaluate changes in the mRNA level with a double comparison: of the studied gene with a control gene between different samples. Reactions were carried out in relative measurement mode (RQ-relative quantification software, Applied Biosystems, USA). For the reactions, the prepared reaction mixtures Synthol (Russia) were used. Reaction conditions: 10 min at 95 ° C (“hot start” to activate DNA polymerase) and then 40 cycles: 15 sec at 95 ° C (denaturation) and 1 min at 60 ° C (annealing with primers and elongation) . Standard curves i.e. curves of CT versus decimal logarithm of the amount of cDNA introduced (log C0) for the studied cDNA sequences and calculation of amplification efficiencies for each sequence were calculated using ABI Prism 7500 SDS Software Version 2.0.3 (Applied Biosystems, United States). Real-time PCR results were analyzed using ABI Prism 7500 SDS Software Version 2.0.3 (Applied Biosystems, USA) in accordance with the recommendations of Applied Biosystems. For the reactions used 10 pcM and 10 pcM primers and linear destructible probe (TaqMan®), respectively. As a control gene, the gene of the ribosomal protein Rpl32 was used. The following pairs of primers and probes were used for amplification:

for the Rpl32 gene: primers Rpl32dir 5′-CCAGCATACAGGCCCAAGATC-3 ′, Rpl32rev 5′-ACGCACTCTGTTGTCGATACC-3 ′, probe - FAM-CGCACCAAGCACTTCATCCGCCAC-BHQ1;

for the Cyp6g1 gene: primers Cyp6g1f 5′-GCGATCCATTGGGCTATAAT-3 ′, Cyp6g1r 5′-CCAATCTCCTGCATAAGGGT-3 ′, probe - FAM-TCGCACCAAGCTGACTCCCG-BHQ1;

for CG1681 gene (GST-thet a ): primers CG1681f5′-TTCGCACCCACTCTCTAGTCAC-3 ′, CG1681r 5′-GCTCGATTGGTTCAGGAAAT-3 ′, probe — FAM-TCAACGAGATGTCGCAGCCACTC-BHQ1;

for the ss gene: primers SSdir 5′-CGAAGGCGACGCAACGG-3 ′, SSrev 5′-GATGCCGCTTTGATGGATTGC-3 ′, probe — FAM-AGCCTGAAGCCGCCGCCCAAG-BHQ1.

In our experiments, probes were synthesized in Syntol (Russia), oligonucleotide primers in Litech (Russia). The analysis of PCR-PB products was carried out on a 2% agarose TAE gel. All amplification products had the expected size.

The results of the analysis of the level of transcription of CG1681, CYP6G1 and ss genes in the analyzed flies are presented in Fig.7.

A comprehensive assessment of the properties of serotonin allows you to attribute it to substances with a radioprotective effect. The reason for this is the significant improvement in the segmentation of the tarsal structures of the extremities of the ss ^aSc adults, which were subjected to x-ray irradiation in combination with the action of serotonin during the larval period of development. Improvement is expressed in an increase in the number of tarsal segments to four to five with one hundred percent survival. Apparently, the reason for this is to increase the level of transcription of the ss gene by 6 times (Fig.7).

The totality of the features of the invention in the form of the use of Drosophil a mel a nog a ster flies combining hypomorphic mutations of the ss and CG5017 genes in its genome to evaluate the test substance for its ability to correct disturbances in the development of adult limbs and transcription of CG1681 and CYP6G1 and caused by radiation ss-genes allows you to obtain a technical result in the form of effective fast directed selection and determination of the properties of substances with toxicoprotective, radioprotective, toxicosensitizing and radiosensitizing possessing properties.

The invention is intended for use in pharmacological firms and research institutes of a medical and biological profile, for laboratory and experimental evaluation of the pharmacological, toxicological and pharmaceutical properties of substances with alleged toxicoprotective, radioprotective, toxicosensitizing and radiosensitizing properties, when studying the properties of substances that can increase or decrease toxicity action of ionizing radiation on a biological object, in order to Senia and recommendations on the application of their findings. The line of flies used in the invention contains mutations of highly conserved genes, leading to developmental and metabolic disorders according to scenarios similar to both a fly and a human. Therefore, it is proposed to use them as a sensitive test system for searching for pharmaceutical agents that can affect the toxic effect of radiation and other exogenous and endogenous factors. Pharmaceutical agents found using the present invention may also be in demand in gerontology, since with increasing age, a person has an increased risk of mosaic occurrence of somatic mutations.

Sources

1. (KR 20120020820 (A) - SCREENING METHOD OF A TOXIC MATERIAL POLLUTING INTERIOR ATMOSPHERE USING DROSOPHILA MELANOGASTER).

2. Jux B. et al. 2011 Journal of Investigative Dermatology 131, 203-210.

3. Dranovsky et al. 2011, Neuron 70, 908-923, June 9, 2011.

4. Dubnau et al., 2003, Current Biology, Vol. 13, 286-296, February 18.

5. Kuzin B.A. et al. 2010. Ontogenesis. 2010.V. 41. No. 5. S.364-370.

Claims (1)

A method of evaluating the pharmacological and toxicological properties of substances - radio, radio and toksikoprotektorov, toksikosensibilizatorov consists in that the medium of the larvae and flies Drosophil a mel a nog a ster wild type and combining its genome ss- hypomorphic mutations and genes CG5017- the test substance is introduced, the larvae or flies are irradiated with ionizing rays in a dose of 1-10 x-rays, the irradiated adults are fixed in ethanol and liquid nitrogen, the viability, limb structures and transcription level of the CG1681, CYP6G1 and ss genes are assessed, p The obtained characteristics of flies grown on a medium containing a test substance and flies grown on a medium not containing a test substance, irradiated and non-irradiated, determine the pharmacological properties of the substance by comparing the viability, the number of tarsal limb segments and the level of transcription of genes CG1681, CYP6G1 and ss in flies of all formed groups.

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